Method for the evolutionary design of biochemical reaction networks

ABSTRACT

The present invention relates to methods for achieving an optimal function of a biochemical reaction network. The methods can be performed in silico using a reconstruction of a biochemical reaction network of a cell and iterative optimization procedures. The methods can further include laboratory culturing steps to confirm and possibly expand the determinations made using the in silico methods, and to produce a cultured cell, or population of cells, with optimal functions. The current invention includes computer systems and computer products including computer-readable program code for performing the in silico steps of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 11/525,380 filed Sep. 22, 2006, now pending; which is a divisionalapplication of U.S. application Ser. No. 09/940,686 filed Aug. 27, 2001,now issued as U.S. Pat. No. 7,127,379; which claims the benefit under 35USC § 119(e) to U.S. Application Ser. No. 60/265,554 filed Jan. 31,2001, now abandoned. The disclosure of each of the prior applications isconsidered part of and is incorporated by reference in the disclosure ofthis application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to biochemical reaction networks andmore specifically to reconstruction of metabolic networks in an organismto obtain optimal desired whole cell properties.

2. Background Information

Genome sequencing and annotation technologies are giving us detailedlists of the molecular components that cells are comprised of, andhigh-throughput technologies are yielding information about how thesecomponents are used. Thus we are approaching the stage where biologicaldesign is possible on a genome scale. It has proven difficult to‘splice’ one gene from one organism into another and produce predictableresults. The primary reason is that every component in a living cell hasbeen honed through a lengthy evolutionary process to ‘fit’ optimallyinto the overall function of the cell. Simply introducing a foreigngene, or deleting an existing gene does not lead to predictable noroptimal results. Methods are needed to a priori predict the consequencesof single or multiple gene deletions or additions on the function of anentire cellular function and force the remaining components to functionin a predetermined manner. Such methods are lacking, although limitedprogress has been made with metabolic function on a cellular scale.

The interest in the redirection of metabolic fluxes for medical andindustrial purposes has existed for some time. As a result of thisinterest, the field of metabolic engineering has been born, and theprimary goal of metabolic engineering is to implement desirablemetabolic behavior in living cells. Advances and applications of severalscientific disciplines including computer technology, genetics, andsystems science lie at the heart of metabolic engineering.

The traditional engineering approach to analysis and design utilizes amathematical or computer model. For metabolism this would require acomputer model that is based on fundamental physicochemical laws andprinciples. The metabolic engineer then hopes that such models can beused to systematically ‘design’ a new and improved living cell. Themethods of recombinant DNA technology should then be applied to achievethe desired cellular designs.

The 25-30 year history of metabolic analysis has demonstrated the needto quantitate systemic aspects of cellular metabolism, (see e.g., FellD., Understanding the control of metabolism, (London, Portland Press)(1996); Heinrich R., et al., Metabolic regulation and mathematicalmodels, Progress in Biophysics and Molecular Biology, 32: 1-82, (1977);Heinrich R. and Schuster S., The regulation of cellular systems, (NewYork, Chapman & Hall), xix, p. 372 (1996); Savageau M. A., Biochemicalsystems analysis. I. Some mathematical properties of the rate law forthe ecomponent enzymatic reactions, J. Theor. Biol. 25(3):365-69(1969)). There are significant incentives to study metabolic dynamics. Aquantitative description of metabolism and the ability to producemetabolic change is not only important to achieve specific therapeuticgoals but has general importance to our understanding of cell biology.Important applications include strain design for the production oftherapeutics and other biochemicals, assessment of the metabolicconsequences of genetic defects, the synthesis of systematic methods tocombat infectious disease, and so forth. Quantitative and systemicanalysis of metabolism is thus of fundamental importance. However, areview in the field has concluded that “despite the recent surge ofinterest in metabolic engineering, a great disparity still existsbetween the power of available molecular biological techniques and theability to rationally analyze biochemical networks” (Stephanopoulos G.,Metabolic engineering. Current Opinions in Biotechnology, 5:196-200(1994)). Although this statement is a few years old, it still basicallyholds true. This conclusion is not surprising for we are competing withmillions of years of natural evolution that achieves the best fitness ofan organism in a given environment.

Although partial gene regulatory networks containing a small number ofreactions have been designed (reviewed in Hasty et al., Computationalstudies of gene regulatory networks: In numero molecular biology,Nature, 2: 268-79 (2001)), the a priori design of biochemical regulatorynetworks, such as metabolic networks with defined performancecharacteristics and their subsequent construction has not been reducedto practice. The primary reason is that reliable detailed kinetic modelscannot be constructed for an entire metabolic network, mainly becausethere are too many kinetic parameters whose numerical values must bedetermined and the detailed kinetic equations are by-and-large unknown.Thus, a priori design of optimal biochemical reaction networks, such asmetabolism, is not possible because predictive kinetic models cannot beachieved. In fact, the values of the kinetic constants change with timedue to mutations and an evolutionary process.

Heretofore it has been impossible to predict the end point ofevolutionary processes as they are expected to be the outcome of theselection from random events. This invention discloses a method thatallows for the a priori calculation of the endpoint of the evolution ofmetabolic networks in a defined environment. Although there are othermathematical modeling methods that are based on optimization principlesin biological systems; i.e. the cybernetic modeling approach (Varner J.and Ramkrishna D., “Mathematical models of metabolic pathways,” Curr.Opin. Biotechnol., 10(2): 146-50, (1999), they are not amenable to thedesign of biological networks due to the number of parameters required.It thus gives the basis for the use of an evolutionary process to createor build such designs.

SUMMARY OF THE INVENTION

The present invention relates to a method for achieving an optimalfunction of a biochemical reaction network in a living cell. Thebiochemical reaction network can be a comprehensive biochemical reactionnetwork, a substantially whole biochemical reaction network, or a wholebiochemical reaction network. The method can be performed in silicousing a reconstruction of a biochemical reaction network of a cell. Themethod can further include laboratory culturing steps to confirm andpossibly expand the determinations made using the in silico steps, andto produce a cultured cell, or population of cells, with optimalfunctions.

The method can be performed by representing a listing of biochemicalreactions in a network in a computer, such as by providing a database ofbiochemical reactions in a network; using optimization methods tocalculate the optimal properties of the network; altering the list ofreactions in the network and re-computing the optimal properties; andrepeating the process described above until the desired performance isreached. The method may further include constructing the genetic makeupof a cell to contain the biochemical reactions which result from theoptimization procedure; placing the cells constructed thereunder inculture under the specified environment; and cultivating the cells for asufficient period of time under conditions to allow the cells to evolveto the determined desired performance.

The biochemical reaction network can be a metabolic network, for examplea regulatory network. In addition, the cell whose genetic makeup isconstructed can be a prokaryotic cell or a eukaryotic cell; such as E.coli, S. cerevisiae, chinese hamster ovary cells, and the like.Furthermore, the genetic makeup of a cell can be constructed by alteringone or more genes in the cell, for example by addition or deletion, orby altering the regulation of a gene through its regulatory components(e.g., promoter, transcription factor binding sites, etc.). In anotheraspect, the invention provides an enriched population of cells producedby the method described above.

In another aspect, the present invention provides a method for achievingoptimal functions of a comprehensive biochemical reaction network in acell by providing a database including biochemical reactions in thenetwork; using optimization methods to calculate the optimal propertiesof the network; receiving a user's selection for altering the reactionsin the network and recomputing the optimal properties; repeatingoptimization until the desired property criterion is met; displaying theresults of the optimization for constructing the genetic makeup of acell so that it contains the biochemical reactions as a result of theoptimization information; culturing the cells constructed under thespecified environment conditions; and cultivating the cells for asufficient period of time so that the cells evolve to the desiredperformance.

The optimization method may be carried out using a computer systemprovided by the present invention. The computer system typicallyincludes a database that provides information regarding one or morebiochemical reaction networks of at least one organism; a user interfacecapable of receiving a selection of one or more biochemical reactionnetworks for optimization and/or comparison, and capable of receiving aselection of a desired performance; and a function for carrying out theoptimization method calculations and recalculations. The computer systemof the present invention can include a function for performingbiochemical reaction network reconstruction. The database can be aninternal database or an external database.

In another aspect the present invention provides a computer programproduct that includes a computer-usable medium having computer-readableprogram code embodied thereon. The program code is capable ofinteracting with the database and effects the following steps within thecomputing system: providing an interface for receiving a selection of adesired performance of the networks; determining the desired optimalproperties, displaying the results of the determination, and alteringthe biochemical reaction network, before recalculating optimalproperties of the biochemical reaction network, and repeating theprocess until a desired optimal function is achieved. Altering thebiochemical reaction network can be performed based on an alterationmanually input by a user, or can be performed automatically by theprogram code. The computer program can further provide an identificationof database entries that are part of a reconstructed biochemicalnetwork, or can perform biochemical reaction network reconstruction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: The acetate uptake rate (AUR in units of mmole/g-DW/hr, g-DW isgram dry weight) versus oxygen uptake rate (OUR, in units ofmmole/g-DW/hr) phenotype phase plane. The in silico defined line ofoptimality (LO) is indicated in the figure. The slope of this line isalso indicated in the figure. The experimental data points are displayedon the figure. The error bars represent a single standard deviation, andthe error bars are displayed for both the acetate and the oxygen uptakerate measurements. A linear regression was performed on the data pointsto define the experimentally reconstructed line of optimality. Thecorrelation coefficient R² value for the curve fit is 0.92. Regions 1 &2 represent distinct non-optimal metabolic phenotypes

FIG. 2: The three-dimensional rendering of the phase surface for growthof E. coli on acetate. The x and y axis represent the same variables asin FIG. 1. The third dimension (the z-dimension) represents the cellulargrowth rate. The z-axis values are in gray scale with the optimal growthrate value quantitatively indicated on the corresponding legend. Theline of optimality (LO) in three-dimensions is indicated. The parametricequation of LO in three-dimensions is indicated in the text. The blacklines define the surface of the metabolic capabilities in thethree-dimensional projection of the flux cone and represent constantvalues of the acetate uptake rate or oxygen uptake rate. Thequantitative effect on cellular growth potential of increasing theacetate uptake rate (without proportional increase in the oxygen uptakerate) can be visualized. The data points are also plotted on thethree-dimensional figure and error bars have been omitted.

FIG. 3: Line of optimality for growth on acetate projected onto a planeformed by the acetate uptake rate and the growth rate. The data pointshave also been projected and a linear regression was performed in thetwo-dimensional plane to experimentally define the line of optimality.The line of optimality is indicated as a gray line and the regressionline as a black line.

FIG. 4: Line of optimality for growth on acetate projected onto a planeformed by the oxygen uptake rate and the growth rate. The data pointshave also been projected and a linear regression was performed in thetwo-dimensional plane to experimentally define the line of optimality.The line of optimality is indicated as a gray line and the regressionline as a black line.

FIG. 5: The succinate uptake rate (mmole/g-DW/hr) versus oxygen uptakerate (mmole/g-DW/hr) represented in the phenotype phase plane. Thelabeled line is the in silico defined line of optimality (LO). Theexperimental data points are displayed on the figure. The error bars aredisplayed for both the succinate and oxygen uptake rate measurements andrepresent a single standard deviation. Cultivations for which acetatewas produced above a threshold of 0.3 mmole/gDW/hr are indicated by opencircles, filled circles identify either no acetate production orproduction below the threshold. The black dotted line represents thelinear regression of the data points with no acetate production.

FIG. 6: The measured acetate production vs. the in silico predictionsfor each point illustrated in FIG. 5. The data points are rank orderedby the magnitude of the succinate uptake rate.

FIG. 7: Three-dimensional phenotype phase plane for E. coli growth onsuccinate. The x and y axis represent the same variables as in FIG. 6.The third dimension (the z-axis) represents the cellular growth rate.The z-axis values are in gray scale with the corresponding legend in thefigure. The demarcation lines separating the colored regions representconstant oxygen and acetate uptake rates, and the quantitative effect ofmoving away from the line of optimality can be visualized. The datapoints are plotted in this three-dimensional figure with the exceptionof the error bars.

FIG. 8: Calculated and experimental values for growth of E. coli K-12 onglucose. The glucose uptake rate, GUR (mmole/gDW/h), and the oxygenuptake rate, OUR (mmole/gDW/h), are shown in the phenotype phase plane.The LO is indicated. Data points are confined to the LO or the acetateoverflow region, where acetate secretion is predicted in silico andexperimentally observed.

FIG. 9: Three-dimensional rendering of growth rates graphed over thephase plane for growth on glucose. The x and y axes represent the samevariables as in FIG. 8. The z-axis represents the cellular growth rate,with color-coded values and the optimal growth rate indicated on thelegend.

FIG. 10: GUR plotted against OUR with experimental values for adaptiveevolution experiments. Data points lie near the LO and in region 2 whereacetate overflow is predicted.

FIG. 11: Three-dimensional rendering of the post-evolutionary 3D growthsurface over the glucose phenotypic phase plane. All data points clustertightly on or near the LO.

FIG. 12: Calculated and experimental values for growth on glycerol. theglycerol uptake rate, GlUR (mmole/gDW/h), and the oxygen uptake rate,OUR (mmole/gDW/h), are shown in the phenotype phase plane. The LO isshown in indicated. The experimental data points are confined to region1, characterized by futile cycles and suboptimal growth rates.

FIG. 13: Three-dimensional rendering of growth rates graphed over theglycerol phenotypic phase plane. The x and y axes represent the samevariables as in FIG. 12. The z-axis represents the cellular growth rate,and the optimal growth rate indicated on the legend. No data points lienear the LO.

FIG. 14: The glycerol uptake rate (GlUR) plotted against the oxygenuptake rate (OUR) with experimental values for adaptive evolutionexperiments. The starting point of evolution is indicated (day 0).Experimental values for the first evolutionary trajectory (E1) areindicated in blue, while values for the second evolutionary trajectory(E2) are indicated in green. In both experiments, the initial strainconverges towards a similar endpoint on the LO, representing optimalgrowth rates.

FIG. 15: Change in growth rate in units or hr⁻¹, with time for adaptiveevolution experiments on glycerol. Both experiments reveal a similaradaptation profile, with increased fitness and a doubling of the growthrate.

FIG. 16: The glycerol-oxygen phenotypic phase plane with experimentalvalues for growth on glycerol after the adaptive evolution experiments.All values cluster tightly on or near the LO. Data represents thetitration of the carbon source and the quantitative effect of movingalong the LO.

FIG. 17 Three-dimensional rendering of the post-evolutionary phasesurface. All data points cluster tightly on or near the LO.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of this invention provides a method to design the propertiesof a large biochemical reaction network. Using a method of this aspectof the present invention, a biochemical reaction network can be designedto a predetermined performance in a specified environment. One aspect ofthe invention includes:

1) using a computer reconstruction of the reaction structure of abiochemical reaction network,

2) using optimization methods to determine the optimal functionalitiesof the reaction network,

3) changing the reaction structure in the computer representation of thenetwork by removing or adding a single or a multitude of genes andrecalculating the optimal properties,

4) using genetic manipulations to get the gene complement in an organismto correspond to the structure of the reaction network whose optimalproperties have been determined through computer simulation, and

5) using extended cultivation under a defined selection pressure toevolve the function of the actual reaction network toward the optimalsolution that was predicted by the computer simulation. The adaptiveevolutionary process will itself determine the best set of kineticparameters to achieve the optimal design. More than one similar set ofparameter values can be determined thorough the evolutionary process.

Using the methods and procedures disclosed herein, a biochemicalreaction network can be designed a priori in a computer. Following thedesign of the reaction network, an evolutionary process is carried outin the laboratory under the appropriate conditions on a geneticallymodified organism or a wild-type strain that corresponds to the networkused for the computer simulations. Organisms may achieve the optimalbehavior in a non-unique fashion—that is there may be equivalent optimalsolutions. Thus the invention involves a non-obvious and non-existingcombination of computer design methods, genetic alteration, andevolutionary process to achieve optimal performance of biochemicalreaction networks within the environment of a living cell.

In another aspect, the present invention relates to a method fordetermining optimal functions of a comprehensive biochemical reactionnetwork in a living cell. The method is used to achieve a desiredperformance of the living cell. The method can be performed byrepresenting a listing of the biochemical reactions in the network in acomputer; using optimization methods to calculate the optimal propertiesof the network; altering the list of reactions in the network andre-computing the optimal properties; and repeating the altering stepuntil the desired performance is met.

In addition to the above steps which are performed in silico, the methodcan further include steps involving culturing a living cell, or apopulation of cells. These steps include constructing the genetic makeupof a cell to contain the biochemical reactions which result fromrepeating the altering step until the desired performance are met;placing the cell constructed thereunder in culture under the specifiedenvironment; and cultivating the cell for a sufficient period of timeand under conditions to allow the cell to evolve to the determineddesired performance.

A biochemical reaction network is an interrelated series of biochemicalreactions that are part of a biochemical pathway or linked biochemicalpathways. Many biochemical reaction networks have been identified suchas metabolic reaction networks, catabolic reaction networks, polypeptideand nucleic acid synthesis reaction networks, amino acid synthesisnetworks, energy metabolism and so forth. Other types of biochemicalreaction networks include regulatory networks including cell signalingnetworks, cell cycle networks, genetic networks involved in regulationof gene expression, such as operon regulatory networks, and actinpolymerization networks that generate portions of the cytoskeleton. Mostof the major cell functions rely on a network of interactive biochemicalreactions.

To practice the present invention, the reaction structure of acomprehensive, preferably substantially whole, or most preferably wholebiochemical reaction network in an organism to be biochemically designedmust be reconstructed for computer simulations. A whole biochemicalreaction network includes all of the biochemical reactions of a cellrelated to a certain biochemical function. For example a whole metabolicreaction network includes essentially all of the biochemical reactionsthat provide the metabolism of a cell. Metabolic reaction networksexemplify a universal biochemical reaction network found in some form inall living cells.

A comprehensive biochemical reaction network is an interrelated group ofbiochemical reactions that effect a detectable property, and that can bemodified in a predictable manner with respect to the effect of suchmodifications on the detectable property in the context of a livingcell. For example, a comprehensive biochemical reaction network caninclude core reactions that effect the yield of a biomolecule producedby the cell, even though the core reactions include only a portion ofthe reactions in the whole biochemical reaction network involved inyield of the biomolecule, provided that computational methods can beused to predict the effect of changes in the core biochemical reactionson the yield in a living cell.

A substantially whole biochemical reaction network is an interrelatedgroup of biochemical reactions that are responsible for a detectableproperty of a living cell. Substantially whole biochemical reactionnetworks include core reactions as well as secondary reactions that havean effect on the detectable property, even though this effect can berelatively minor. Changes in substantially whole biochemical reactionnetworks can be predicted using computational methods. The presentinvention can also utilize the majority of reactions in a wholebiochemical reaction network, rather than a comprehensive, substantiallywhole, or whole biochemical reaction network.

Optimal properties, also referred to herein as optimal functions,determined using the methods of the current invention include, forexample, glycerol uptake rate, oxygen uptake rate, growth rate,sporulation occurrence and/or rates, rates of scouring of rare elementsunder nutritionally poor conditions, biomass, and yields of biomoleculessuch as proteins, carbohydrates, antibiotics, vitamins, amino acids,fermentation products, such as lactate production. Optimal propertiesalso include, for example, yields of chiral compounds and other lowmolecular weight compounds. Optimal properties also include, forexample, the maximal internal yields of key co-factors, such as energycarrying ATP or redox carrying NADPH and NADH. Optimal properties canalso be defined by a cellular engineer to include properties such asflux rates through key reactions in the biochemical reaction network.The current invention allows an optimal performance related to one ormore of these properties to be achieved. For example, the methods allowa specific desired growth rate or specific desired yield to be achieved.

Typically for the methods of the current invention, the biochemicalreactions of a reconstructed biochemical reaction network arerepresented in a computer. This representation can include a listing ofthe biochemical reactions in the reconstructed biochemical reactionnetwork. The listing can be represented in a computer database, forexample as a series of tables of a relational database, so that it canbe interfaced with computer algorithms that represent network simulationand calculation of optimality properties.

The biochemical network reconstruction must be of high quality for thepresent invention. The process of high quality biochemical reactionnetwork, specifically metabolic reaction network, reconstruction hasbeen established M. W. Covert, C. H. Schilling, I. Famili, J. S.Edwards, I. I. Goryanin, E. Selkov, and B. O. Palsson, “Metabolicmodeling of microbial stains in silico,” Trends in Biochemical Sciences,26: 179-186 (2001); Edwards J., and Palsson, B, Metabolic flux balanceanalysis and the in silico analysis of Escherichia coli K-12 genedeletions, BMC Structural Biology, 1(2) (2000a); Edwards J. S., andPalsson, B, O., Systemic properties of the Haemophilus influenzae Rdmetabolic genotype, Journal of Biological Chemistry, 274(25):17410-16,(1999); Karp P. D. et al., HinCyc: A knowledge base of the completegenome and metabolic pathways of H. influenzae, ISMB 4:116-24, (1996);Karp P. D. et al., The EcoCyc and MetaCyc databases, Nucleic. Acids Res.28(1):56-59 (2000); Ogata et al., KEGG: Kyoto encyclopedia of genes andgenomes, Nucleic Acids Res. 27(1):29-34 (1999); Schilling C. H. andPalsson B. O., Assessment of the metabolic capabilities of Haemophilusinfluenzae Rd through a genome-scale pathway analysis, J. Theor. Biol.,203(3): 249-83 (2000); Selkov E. Jr. et al., MPW: the metabolic pathwaysdatabase, Nucleic Acids Res., 26(1): 43-45 (1998); Selkov E. et al., Areconstruction of the metabolism of Methanococcus jannaschii fromsequence data. Gene 197(1-2):GC 11-26 (1997)). This process involves theuse of annotated genome sequences, and biochemical and physiologicaldata. These annotated genome sequences and biochemical and physiologicaldata can be found in one or more internal or external databases, such asthose described in detail in the discussion of the computer systems ofthe current invention below. Careful analysis of the reconstructednetwork is needed to reconcile all the data sources used. Similarmethods can be used for the reconstruction of other biochemical reactionnetworks.

A method of this aspect of the present invention then uses thereconstructed comprehensive, substantially whole, or whole biochemicalreaction network to determine optimal properties of the comprehensive,substantially whole, or whole biochemical reaction network underspecified and varying environmental conditions. This determinationallows the design of a biochemical reaction network that achieves adesired performance in a specified environment. This in turn, can becombined with steps for constructing the genetic makeup of a cell andcultivating the cell, described below, to provide a method fordeveloping a recombinant cell, or a population of cells, that achievesthe desired performance.

Optimal properties of the comprehensive, substantially whole, or wholebiochemical reaction network under a series of specified environmentscan be determined using computational methods known as optimizationmethods. Optimization methods are known in the art (see e.g., Edwardsand Palsson (1999)). The optimization methods used in the methods of thecurrent invention can, for example and not intended to be limiting,utilize a combination of flux balance analysis (FBA), phase planeanalysis (PhPP), and a determination of a Line of Optimality (LO), asdescribed in further detail below.

The reconstructed metabolic network can then be used to performquantitative simulations of the metabolic flux distribution in a steadystate using established methods (Bonarius et al., Flux analysis ofunderdetermined metabolic networks: The quest for the missingconstraints, Trends in Biotechnology, 15(8): 308-14 (1997); Edwards J.S., et al., Metabolic flux Balance Analysis, In: (Lee S. Y., PapoutsakisE. T., eds.) Metabolic Engineering: Marcel Deker. P 13-57 (1999); VarmaA. and Palsson B. O., Metabolic flux balancing: Basic concepts,Scientific and practical use, Bio/Technology 12:994-98 (1994a)).Computer simulations of the metabolic network can be performed under anyconditions. Furthermore, any reaction list can be simulated in acomputer by changing the parameters describing the environment and thecontents of the reaction list.

The metabolic capabilities of a reconstructed metabolic network can beassessed using the established method of flux balance analysis (FBA)(Bonarius et al., (1997); Edwards et al. (1999); Varma and Palsson(1994a)). FBA is based on the conservation of mass in the metabolicnetwork in a steady state and capacity constraints (maximal fluxesthrough the reactions) on individual reactions in the network.Additionally, experimentally determined strain specific parameters arealso required, the biomass composition (Pramanik J. and Keasling J. D.,Stoichiometric model of Escherichia coli metabolism: Incorporation ofgrowth-rate dependent biomass composition and mechanistic energyrequirements, Biotechnology and Bioengineering, 56(4): 398-421 (1997))and the maintenance requirements (Varma A. and Palsson B. O.,Stoichiometric flux balance models quantitatively predict growth andmetabolic by-product secretion in wild-type Escherichia coli W3110.Applied and Environmental Microbiology, 60(10): 3724-31 (1994b)). Thesefactors are then used to calculate the flux distribution through thereconstructed metabolic network.

More specifically, the definition of these factors leads mathematicallyto a closed solution space to the equations in which all feasiblesolutions lie. There are thus many possible solutions (fluxdistributions) to the problem. The ‘best’ or optimal solution within theset of all allowable solutions can then be determined using optimizationprocedures and a stated objective. The optimization procedure used hasbeen linear programming and the objective is the optimal use of thebiochemical reaction network to produce all biomass componentssimultaneously. These optimization procedures are established and havebeen published (Varma and Palsson (1994a); Bonarious (1997); and Edwardset al. (1999)). The comparison of the calculated behavior based on theoptimal growth objective to the experimental data is favorable in themajority of cases (Varma (1994b); Edwards J. S., Ibarra R. U., andPalsson B. O.(herein incorporated by reference), In silico predictionsof Escherichia coli metabolic capabilities are consistent withexperimental data, Nat Biotechnol., 19(2): 125-30 (2001a); and Edwards,Ramakrishna, and Palsson, Characterizing phenotypic plasticity: Aphenotype phase plane analysis, Biotech Bioeng, In Press, (2001b)). Inother words, these solution confinement and optimization procedures leadto a prediction of the optimal uses of a biochemical reaction network tosupport cellular growth and for pre-evolved strains give a good estimateof actual biological function.

The use of alternate survival objectives, such as sporulation, andscouring of rare elements under nutritionally poor conditions, has notbeen described. Competition and evolution under these conditions canalso be used to define and generate optimal network functions.

These procedures lead to the calculation of optimal function under asingle growth condition. This is very limiting and a method to analyze alarge number of growth conditions is needed.

As stated above, all steady state metabolic flux distributions aremathematically confined to the solution space defined for a givenreconstructed metabolic network, where each solution in the solutionspace corresponds to a particular flux distribution through the networkor a particular metabolic phenotype (Edwards and Palsson (1999)). Undera single specified growth condition, the optimal metabolic fluxdistribution in the cone can be determined using linear programming (LP)or other related approaches for calculating optimal solutions of suchproblems. If the constraints vary, the shape of the cone changes and theoptimal flux vector may qualitatively change. Phenotype Phase Plane(PhPP) analysis considers all possible variations in two or moreconstraining environmental variables. This method is now disclosed.

Uptake rates of two nutrients (such as the carbon substrate and oxygen)can be defined as two axes on an (x,y)-plane, and the optimal fluxdistribution can be calculated for all points in this plane using theprocedures described above. When this procedure is implemented for areconstructed metabolic network that is biologically realistic, we findthat there are a finite number of qualitatively different optimalmetabolic flux maps, or metabolic phenotypes, present in such a plane.The demarcations on the phase plane can be defined by using shadowprices of linear optimization (Chvatal V., Linear Programming, (NewYork: W. H. Freeman and Co.) (1983)). The procedure described leads tothe definition of distinct regions, or “phases”, in the (x,y)-plane, forwhich the optimal use of the metabolic network is qualitativelydifferent. Each phase can be designated as Pnx,y, where P represents aparticular phenotype or flux distribution, n is the number arbitrarilyassigned to the demarcated region for this phenotype, and the two uptakerates form the axis of the plane.

The phase planes can be constructed by calculating the shadow pricesthroughout the two-parameter space, and lines are drawn to demarcateregions of constant shadow prices. The shadow prices define theintrinsic value of each metabolite toward the objective function(Chvatal (1983)). The shadow prices are either negative, zero, orpositive, depending on the value of the metabolite to optimizing growthunder a given environmental condition, as represented by particularnumerical values of the uptake rates represented by the x and y axes.When shadow prices become zero as the values of the uptake rates arechanged there is a qualitative shift in the optimal metabolic map. Thiscriterion defines the lines in the PhPP.

The line of optimality (LO) is defined as the line representing theoptimal relation between the two uptake fluxes corresponding to the axesof the PhPP. For aerobics, this line can be interpreted as the optimaloxygen uptake for the complete oxidation of the substrate in order tosupport the maximal biomass yield.

The metabolic reconstruction and phenotype phase plane analysisprocedures are then used to predict the conditions under which thedesired metabolic behavior, for example maximum uptake rates, will beoptimal. In other words, metabolic reconstruction and PhPP are used todetermine optimal performance. The maximal uptake rates lead to thedefinition of a finite rectangular region in the phase plane. Theoptimal growth condition within this rectangle will then be thepredicted outcome of an evolutionary process within the givenconstraints.

Using the optimization procedure, the properties of the correspondingactual biochemical reaction network may not be optimal or the same asdesired from a practical standpoint. The simulated reconstructed networkand its synthesis in an organism may not display the optimal solutiondesired, also referred to herein as the desired optimal performance ordesired optimal function. Lack of optimality may be due to the factthat:

1. The natural organism with an intact network has never experienced theenvironmental conditions of interest and never undergone growthcompetition and selection in this environment, or

2. The man made network is perturbed from its evolutionarily determinedoptimal state by genetic manipulations, through the deletion/addition ofa new reaction from/to the network.

The in silico methods of the current invention are designed to resolvethis second cause of lack of optimality, by altering the reactions inthe network until a desired performance is achieved. Then culturingmethods, which can be included in the method of the current invention asdescribed in further detail below, can be used to resolve the firstcause of the lack of optimality related to growth competition andselection.

As mentioned above, after calculation of the optimal properties, ametabolic engineer can alter the reaction list in the network, or analgorithm can be developed that automatically alters one or morereactions in the reaction list, to achieve a desired performance. Afteralteration of the biochemical list, optimal properties of this networkunder given environmental conditions can be calculated. This is aniterative design procedure that may require many different versions ofthe reaction list until the desired performance is achieved. The desiredperformance is a qualitative characteristic or quantitative value for aproperty calculated using an optimization procedure. Many properties forwhich a desired performance can be achieved are known in the art. Forexample, a desired performance can be a desired growth rate or a desiredyield of a biomolecule such as an enzyme or an antibiotic.

The optimization method may be carried out using a computer systemprovided by the present invention. The computer system typicallyincludes a database that provides information regarding one or morebiochemical reaction networks of at least one organism; a user interfacecapable of receiving a selection of one or more biochemical reactionnetworks for optimization and/or comparison, and capable of receiving aselection of a desired performance; and a function for carrying out theoptimization method calculations and recalculations. Furthermore, thecomputer system of the present invention may include a function forperforming biochemical reaction network reconstruction describedhereinabove.

The computer system can be a stand-alone computer or a conventionalnetwork system including a client/server environment and one or moredatabase servers. A number of conventional network systems, including alocal area network (LAN) or a wide area network (WAN), are known in theart. Additionally, client/server environments, database servers, andnetworks are well documented in the technical, trade, and patentliterature. For example, the database server can run on an operatingsystem such as UNIX, running a relational database management system, aWorld Wide Web application, and a World Wide Web Server.

Typically, the database of the computer system of the present inventionincludes information regarding biochemical reactions in a comprehensivebiochemical reaction network, a substantially whole biochemical reactionnetwork, or a whole biochemical reaction network. This information caninclude identification of biomolecular reactants, products, cofactors,enzymes, rates of reactions coenzymes, etc. involved in at least some ofthe biochemical reactions of the network. This information can includethe stoichiometric coefficients that indicate the number of molecules ofa compound that participates in a particular biochemical reaction. Thisinformation can include any and all isozymes that are found in theorganism. This information can include all the related biochemicalreactions that can be catalyzed by a single enzyme. This information caninclude the formation of oligomeric enzyme complexes, that is when manydifferent protein molecules must non-covalently associate to form acomplex that can carry out the chemical reactions. This information caninclude the location of the enzyme that carries out the reaction, (i.e.if it is in a membrane, in the cytoplasm, or inside an organelle such asthe mitochondria). The information can also include experimentallyderived or calculated rates of reactions under various conditions,biomass compositions, and maintenance requirements. This information caninclude non-mechanistic growth and non-growth associated maintenancerequirements. This information can include mechanistic maintenanceinformation such as inefficiency in protein synthesis. This informationcan include data derived from genome-scale mRNA or protein expressionprofiles. This information can include data on operon or regulatorystructures that are associated with the expression of a particular gene.This information can include sequence variations that reflect changes inenzyme properties. This information can include a condition-dependentinclusion of a biochemical reaction, depending for instance if a gene isnot expressed under the conditions of interest. Where the biochemicalreaction network is a metabolic reaction network, the information forexample can include known consumption rates, by-product productionrates, and uptake rates.

The information in the database may include biomolecular sequenceinformation regarding biomolecules involved in the biochemical reactionsof the biochemical reaction network, for example information regardingmultiple biomolecular sequences such as genomic sequences. At least someof the genomic sequences can represent open reading frames located alongone or more contiguous sequences on each of the genomes of the one ormore organisms. The information regarding biochemical reaction networkscan include information identifying those biochemical reaction networksto which a biomolecular sequence plays a role and the specific reactionsin the biochemical reaction network involving the biomolecule.

The database can include any type of biological sequence informationthat pertains to biochemical reactions. For example, the database can bea nucleic acid sequence database, including ESTs and/or more preferablyfull-length sequences, or an amino acid sequence database. The databasepreferably provides information about a comprehensive, substantiallywhole, or whole biochemical reaction network. For example, the databasecan provide information regarding a whole metabolic reaction network.The database can provide nucleic acid and/or amino acid sequences of anentire genome of an organism.

The database can include biochemical and sequence information from anyliving organism and can be divided into two parts, one for storingsequences and the other for storing information regarding the sequences.For example, the database can provide biochemical reaction informationand sequence information for animals (e.g., human, primate, rodent,amphibian, insect, etc), plants, or microbes. The database is preferablyannotated, such as with information regarding the function, especiallythe biochemical function, of the biomolecules of the database. Theannotations can include information obtained from published reportsstudying the biochemistry of the biomolecules of the database, such asspecific reactions to which a biomolecule is involved, whether thebiomolecule is or encodes an enzyme, whether the sequence is a wild-typesequence, etc.

The annotations and sequences of the database provide sufficientinformation for a selected biochemical genotype of an organism to beidentified. A biochemical genotype is a grouping of all the nucleic acidor amino acid sequences in a selected biochemical process of anorganism. For example, a metabolic genotype is a grouping of all thenucleic acid and/or amino acid sequences of proteins involved inmetabolism. Methods for identifying metabolic genotypes have beendescribed in the literature (see e.g. Edwards and Palsson 1999).

The database can be a flat file database or a relational database. Thedatabase can be an internal database, or an external database that isaccessible to users, for example a public biological sequence database,such as Genbank or Genpept. An internal database is a databasemaintained as a private database, typically maintained behind afirewall, by an enterprise. An external database is located outside aninternal database, and is typically maintained by a different entitythan an internal database. A number of external public biologicalsequence databases are available and can be used with the currentinvention. For example, many of the biological sequence databasesavailable from the National Center for Biological Information (NCBI),part of the National Library of Medicine, can be used with the currentinvention. Other examples of external databases include the Blocksdatabase maintained by the Fred Hutchinson Cancer Research Center inSeattle, and the Swiss-Prot site maintained by the University of Geneva.Additionally, the external databases can include a database providinginformation regarding biochemical reactions, including databases ofpublished literature references describing and analyzing biochemicalreactions. Where a database included in the computer systems of thepresent invention is a public computer database that does not identifyinformation that is relevant for a particular biochemical reactionnetwork, the computer system either includes a function for performingbiochemical reaction network reconstruction, or includes identificationof the database entries that pertain to a particular biochemicalreaction network. Additionally, there are several databases withbiochemical pathway information, these databases include, fornon-limiting example, EcoCyc, KEGG, WIT, and EMP. These databases can beused to provide the information to reconstruct the metabolic models.

In addition to the database discussed above, the computer system of thepresent invention includes a user interface capable of receiving aselection of one or more biochemical reaction networks for optimizationand/or comparison, and capable of receiving a selection of an optimalperformance. The interface can be a graphic user interface whereselections are made using a series of menus, dialog boxes, and/orselectable buttons, for example. The interface typically takes a userthrough a series of screens beginning with a main screen. The userinterface can include links that a user may select to access additionalinformation relating to a biochemical reaction network.

The function of the computer system of the present invention thatcarries out the optimization methods typically includes a processingunit that executes a computer program product, itself representinganother aspect of the invention, that includes a computer-readableprogram code embodied on a computer-usable medium and present in amemory function connected to the processing unit. The memory functioncan be, for example, a disk drive, Random Access Memory, Read OnlyMemory, or Flash Memory.

The computer program product that is read and executed by the processingunit of the computer system of the present invention, includes acomputer-readable program code embodied on a computer-usable medium. Theprogram code is capable of interacting with the database and effects thefollowing steps within the computing system: providing an interface forreceiving a selection of a desired performance of the networks;determining the desired optimal properties, displaying the results ofthe determination, and altering the biochemical reaction network, beforerecalculating optimal properties of the biochemical reaction network,and repeating the process until a desired performance is achieved.Altering the biochemical reaction network can be performed based on analteration identified by a user, or can be performed automatically bythe program code. The computer program can further provide anidentification of database entries that are part of a reconstructedbiochemical network, or can perform biochemical reaction networkreconstruction. Furthermore, the computer program of the presentinvention can provide a function for comparing biochemical reactionnetworks to identify differences in components and properties.

The computer-readable program code can be generated using any well-knowncompiler that is compatible with a programming language used to writesoftware for carrying out the methods of the current invention. Manyprogramming languages are known that can be used to write software toperform the methods of the current invention.

As mentioned above, a method of this aspect of the invention can furtherinclude steps that involve adaptive evolution of a cultured strain toachieve the desired performance. Virtually any cell can be used with themethods of the present invention including, for example, a prokaryoticcell, or a eukaryotic cell such as a fungal cell or an animal cellincluding a cell of an animal cell line. However, a biochemical reactionnetwork of the cell, or the cell of a closely related organism, must besufficiently characterized to allow a high quality reconstruction of thecomprehensive, substantially whole, and/or whole biochemical reactionnetwork in a computer. Preferably, essentially the entire genome of theorganism has been sequenced and genes encoding biomolecules, typicallyproteins, involved in the biochemical reaction network have beenidentified.

The genetic makeup of a cell can be constructed to contain thebiochemical reactions that meet the desired performance to produce acell with a potential to meet the desired performance. This can beachieved using the indigenous list of reactions in the cell and byadding and subtracting reactions from this list using geneticmanipulations to achieve the reaction list capable of achieving thedesired performance criteria, identified by the steps performed insilico described above. For example, reactions can be added orsubtracted from the list by adding, changing, or deleting all orportions of one or more genes encoding one or more biomolecules involvedin the reaction, for example by adding, changing, or deleting proteincoding regions of one or more genes or by adding, changing, or deletingregulatory regions of one or more genes. In addition, for example,reactions can be added or subtracted from the list by alteringexpression of regulatory components (e.g., transcription factors) thateffect the expression of one or more biomolecules involved in one ormore reactions of the reaction list. The resulting engineered cell mayor may not display the optimal properties calculated ahead of time bythe in silico methods using the iterative optimization proceduredescribed above.

Many methods exist in the art that describe the genetic manipulations ofcells (see e.g. Datsenko K. A. and Wanner B. L., One-step inactivationof chromosomal genes in Escherichia coli K-12 using PCR products, Proc.Natl. Acad. Sci. U.S.A., 97(12):6640-45 (2000); Link et al., Methods forgenerating precise deletions and insertions in the genome of wild-typeEscherichia coli: Application to open reading frame characterization, J.of Bacteriology, 179:6228-37 (1997)). Any of the existing geneticmanipulation procedures can be used to practice the invention disclosed.

After the cell has been constructed to have a potential to meet thedesired performance, it is placed in culture under a specifiedenvironment. The specified environment is determined during theoptimization procedure. That is, the optimization procedure calculatesproperties of the network under various environments, as describedabove, and identifies the specified environment in which the desiredperformance is achieved.

The cells are cultured for a sufficient period of time and underconditions to allow the cells to evolve to the desired performance. Thatis, adaptive evolution of natural or engineered strains is carried outas guided by the general optimization methods or procedures. Naturalstrains that have not experienced a particular environment orgenetically altered strains can be analyzed by the networkreconstruction and optimization procedures disclosed above. Thesestrains can then be put under a selection pressure consistent with thedesired function of the organisms and evolved towards the predeterminedperformance characteristics. The cells may achieve the desiredperformance without additional adaptive evolution. That is, thesufficient period of time for culturing the cells, may be immediatelyafter the genetic makeup of the cells is constructed using the methodsof the present invention without the need for further adaptiveevolution.

In other words, Extended cultivation of a non-optimal or non-evolvedstrain can be performed to optimize or evolve the metabolic networktoward the optimal solution that is achievable under the definedenvironmental conditions. The practice of this evolutionary processrequires on the order of weeks to years to optimize a metabolic networkdepending on how far it is from the optimal conditions at the beginningof the evolutionary process, and how difficult it is to achieve thenecessary changes through random mutation and shifts in regulation ofgene expression. This process can be accelerated by the use of chemicalmutagens and/or radiation. Additionally, the process is accelerated bygenetically altering the living cell so that it contains the biochemicalreactants determined by the in silico method described above, thatachieve a desired performance.

Methods are known in the art for culturing cells under specifiedenvironmental conditions. For example, if the cell is E. coli, and thedesired performance is a desired growth rate, the procedure set outbelow can be used. This procedure can be readily adapted for use withother bacterial cells and/or other performance criteria as is known inthe art. Additionally, the procedures can be readily developed for usewith other cell types such as animal cells. For example, the methods canbe readily adapted for use with other culturing systems, such as largescales systems in which cells adhere to a culturing vessel. Theculturing methods may be adapted for high-throughput analysis, as knownin the art.

If a strain needs to be directionally evolved to achieve the desiredperformance, then following the construction of the metabolic reactionnetwork in the chosen host strain, the cells are typically stored frozenat −80° C. with 30% glycerol. For each adaptive evolutionary process,frozen stocks are plated on LB agar and grown overnight at 37° C. Fromthe plate, individual colonies can be identified that arose from asingle cell. An individual colony can be used to inoculate a liquidculture, known as a pre-culture. Pre-cultures inoculated from a singlecolony of the respective strain are grown overnight in the definedmedium for the subsequent evolutionary process. A pre-culture sample istaken the following day, typically at mid-log phase (in the middle oflogarithmic growth) of growth to inoculate the culture conditions thatdefine the environment that the adaptive evolution is to take place.Batch bioreactors or other suitable culture vessels are then initiated.This, typically would be done at 250 mL volumes in micro-carrier spinnerflasks inside a temperature controlled incubator on top of a magneticstir plate, set at suitable—typically high—speed to ensure sufficientaeration and at the optimal growth temperature (37° C. for wildtype E.coli) for any given strain. Other frequently used cultivation proceduresknown in the art can also be used.

After a suitable time period, typically the following day for E. coli(before the culture reaches stationary phase), an aliquot of the culturenow in mid-log phase is serially transferred to a new spinner flaskcontaining fresh medium. If the culture is being optimized for growthrate, stationary phase must be avoided to ensure that the selectioncriterion is growth rate. Then serial transfers are performed at fixedtime intervals (typically every 24 hours depending on the growth rate)at mid-log phase and the volume of the inoculum into the new culturevessel is adjusted accordingly based on the increase in growth rate.

Growth rate is thus monitored frequently, typically on a daily basis inorder to determine the proper volume of the inoculum to use for the nextserial transfer. This serial cultivation process is repeatedsufficiently often to allow the cells to evolve towards its optimalachievable growth under the conditions specified through the mediumcomposition.

The growth and metabolic behavior is monitored during the adaptiveevolutionary process to determine how the population is evolving overtime. At fixed time intervals typically every few days, the culture istested for metabolic and growth behavior, by measuring the oxygen uptakerate, substrate uptake rate and the growth rate. The results are thenplotted as a data point on the phenotype phase plane. Movement of the sodetermined data point towards the line of optimality would indicateevolution towards optimal growth behavior. These measurements of themembrane transport fluxes along with the growth rate are repeated untilwe observe that the cells are operating their metabolic network suchthat the data points lie at the optimal conditions. The evolutionaryprocess can then be continued until there is no further increase in theoptimal performance, i.e., growth rate. If no further change is observedthen we have achieved the maximal growth rate for the given conditions.

Byproduct secretion can be monitored by HPLC or other suitable methodsof analytical chemistry to assess changes in metabolism that areimplicated in the evolution towards optimal growth behavior. For thesestudies it is also imperative to determine a correlation of dry weightvs optical density for the evolved strain since this will be differentfrom the wildtype. In addition to monitoring the growth rate and steadystate growth, the cultures are inspected for any signs of possiblecontamination or co-evolution with a mutant subpopulation-aliquots foreach day of culture are kept refrigerated as a backup in the event ofany contamination, and the phenotype of the culture is ascertained byplating samples of the culture and inspecting for any differences incolony morphology or different mutants. On a daily basis, the opticaldensity of the culture, time of inoculation, inoculum volume, growthrate, and any signs of contamination, are logged. Samples are alsofrozen at −80° C. in 30% glycerol for each day of culture for anypossible further use.

After cells produced using the methods of the current invention evolveto achieve optimal performance, they may be further characterized. Forexample, a characterization can be made of the biochemical reactionnetwork(s) of the cell. This characterization can be used to compare theproperties, including components, of the biochemical reaction network(s)in the living cells to those predicted using the in silico methods.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLE 1 Detailed Methods for Determination of Optimal Function andEvolution for E. Coli

This example provides cultivation procedures that can be used todetermine the optimality of strain performance and to carry out adaptiveevolutionary processes.

Growth behavior of E. coli is determined by the following standardprocedures. Growth is carried out in M9 minimal media (Maniatis et al.,Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor, N.Y., ColdSpring Harbor Laboratory, 545 (1982)) with the addition of the carbonsource (Table 1). Cellular growth rate is varied by changing theenvironmental conditions, i.e. by changing the carbon sourceconcentration approximately ranging between 0.05-4 g/L, temperature(27.5° C. to 37° C.), and oxygen (0-100% saturation relative to air).Batch cultures are set up in bioreactors at volumes of 250 mL in 500 mLflask with aeration. For these cultures the oxygen uptake rate (OUR) ismonitored online, by either measuring the mass transfer coefficient foroxygen (k₁a), by using an off-gas analyzer, or by monitoring the oxygentension in a respirometer chamber using known methods in the art. Thetemperature is controlled using a circulating water bath (Haake, Berlin,Germany). All measurements and data analysis are restricted to theexponential phase of growth. The biomass and the concentration of thesubstrate and metabolic by-products in the media are monitoredthroughout the experiment using methods known in the art. Cellulargrowth is monitored by measuring the optical density (OD) at 600 nm and420 nm and by cell counts. OD to cellular dry weight correlations aredetermined by two different methods; (1) 50 mL samples of culture arespun down and are dried at 75° C. to a constant weight, and (2) 25 ml(taken throughout the culture) samples are filtered through a 0.45 umfilter and dried to a constant weight. The concentration of metabolitesin the culture media is determined by HPLC. An aminex HPX-87H ionexchange carbohydrate-organic acid column (@66° C.) is used withdegassed 5 mM H₂SO₄ as the mobile phase and UV detection. Enzymaticassays are also used to determine the substrate uptake rate andby-product secretion rates. The dissolved oxygen in the culture ismonitored using a polarographic dissolved oxygen probe. Oxygenconsumption is measured by three different methodologies; (1) passingthe effluent gas through a 1440C Servomex oxygen analyzer (Servomex Co.,Inc. Norwood, Mass.), (2) calculated from the dissolved oxygen readingand k₁a measurements, and (3) in a respirometer chamber in a separate 50ml flask. All three methods used for measuring the oxygen uptake rategive similar and reproducible results.

The cultivation procedures provided in this Example can be used todetermine the optimality of strain performance and to carry out adaptiveevolution.

TABLE I M9 Minimal Media (Per liter) 5 × M9 Salts 200 mL 1M MgSO₄ 2 mL20% Solution of Carbon Source 20 mL 1M CaCl 0.1 mL 5 × M9 Salts (Perliter) Na₂HPO₄*7H₂0 64 g KH₂PO₄ 15 g NaCl 2.5 g NH₄Cl 5.0 g

EXAMPLE 2 Calculation of Optimality Properties and Phenotypic PhasePlanes

This example shows how we calculate the optimality properties of thereconstructed network and how such results are represented on aphenotypic phase plane.

The capabilities of a metabolic network can be assessed using fluxbalance analysis (FBA) (Bonarius et al., (1997); Edwards et al., (1999);Varma et al., (1994a); Varma et al., (1994b)). FBA is primarily based onthe conservation of mass in the metabolic network. The conservationrequirement is implemented by stoichiometric balance equations; thus,FBA relies on the stoichiometric characteristics of the metabolicnetwork.

The flux balance equation is Sv=b^(v), where S is the stoichiometricmatrix, the vector v defines the metabolic fluxes, and b^(v) isnominally zero—thus, enforcing simultaneous mass, energy, and redoxbalance constraints through a set of mass balances. Variations of theb^(v) vector from zero were used in the shadow price analysis (discussedbelow). In the E. coli metabolic network, the number of metabolic fluxeswas greater than the number of mass balances, thus leading to aplurality of feasible flux distributions that lie in the null space ofthe matrix S. Additional constraints were also placed on the feasiblevalue of each flux in the metabolic network (α_(i)≦ν_(i)≦β_(i)). Theseconstraints were utilized to define the reversibility of the internalreactions and to set the uptake rate for the transport reactions. Thetransport of inorganic phosphate, ammonia, carbon dioxide, sulfate,potassium, and sodium were unrestrained; whereas the uptake of thecarbon source and oxygen were restrained as specified. All metabolicby-products (i.e. acetate, ethanol, formate, pyruvate, succinate,lactate, etc) which are known be transported out of the cell were alwaysallowed to leave the metabolic system. In this analysis, α_(i) for theinternal fluxes was set to zero for all irreversible fluxes and allreversible fluxes were unbounded in the forward and reverse direction(the reversibility of each reaction is defined on the website ofsupplementary information). The intersection of the null space, andregion defined by the linear inequalities, defined the capabilities ofthe metabolic network and has been referred to as the flux cone(Schilling et al., 1999).

The determination of the optimal metabolic flux distribution that lieswithin the flux cone was formulated as a linear programming (LP)problem, in which the solution that minimizes a particular metabolicobjective was identified (Bonarius, H. P J. et al., Metabolic fluxanalysis of hybridoma cells in different culture media using massbalances, Biotechnology and Bioengineering 50: 299-318 (1996); Edwardset al., (1999); Pramanik et al., (1997); Varma et al., (1994a); Varma etal., (1994b)). The growth flux was defined as the objective. The growthflux was defined as a metabolic flux utilizing the biosyntheticprecursors, X_(m), in the appropriate ratios,

${{\sum\limits_{{all}\mspace{14mu} m}^{\;}\; {d_{m} \cdot X_{m}}}\overset{v_{growth}}{\rightarrow}{Biomass}},$

where d_(m) is the biomass fraction of metabolite X_(m). The biomasscomposition was defined based on the literature (Neidhardt, F C, Ed.,Escherichia coli and Salmonella: cellular and molecular biology (ASMPress, Washington, D.C., 1996); Neidhardt, F C, Umbarger, H E, inEscherichia coli and Salmonella: cellular and molecular biology F. C.Neidhardt, Ed. (ASM Press, Washington, D.C., 1996), vol. 1, pp. 13-16(1996)).

All steady state metabolic flux distributions are mathematicallyconfined to the flux cone defined for the given metabolic map, whereeach solution in the flux cone corresponds to a particular internal fluxdistribution or a particular metabolic phenotype (Varma et al., (1994a);Varma et al., (1994b)). Under specified growth conditions, the optimalphenotype in the cone can be determined using linear programming (LP).If the constraints vary, the shape of the cone changes and the optimalflux vector may qualitatively change; for example, inactive fluxes maybe activated and vice versa. The phase plane analysis is developed toconsider all possible variations in two constraining environmentalvariables.

Defining Phenotype Phase Planes (PhPPs): Uptake rates of two nutrients(such as the carbon substrate and oxygen) were defined as two axes on an(x,y)-plane, and the optimal flux distribution was calculated for allpoints in this plane. There are a finite number of qualitativelydifferent optimal metabolic flux maps, or metabolic phenotypes, presentin such a plane. The demarcations on the phase plane were defined by ashadow price analysis (Varma, A, Boesch, B W, Palsson, B O.,Stoichiometric interpretation of Escherichia coli glucose catabolismunder various oxygenation rates, Applied and Environmental Microbiology59, 2465-73 (1993); Varma, A, Palsson, B O., Metabolic capabilities ofEscherichia coli: II. Optimal growth patterns. Journal of TheoreticalBiology 165, 503-522 (1993)). This procedure led to the definition ofdistinct regions, or “phases”, in the plane, for which the optimal useof the metabolic pathways was qualitatively different. Each phase waswritten as Pn_(x,y), where P represents phenotype, n is the number ofthe demarcated region for this phenotype (as shown in the correspondingFIG. 1), and x,y the two uptake rates on the axis of the plane.

Calculating the Phase Plane: The phase planes were constructed bycalculating the shadow prices throughout the two-parameter space, andlines were drawn to demarcate regions of constant shadow prices. Theshadow prices defined the intrinsic value of each metabolite toward theobjective function. Changes in shadow prices were used to interpret ofmetabolic behavior.

Mathematically, the shadow prices are defined as,

$\begin{matrix}{\gamma_{i} = {- \frac{Z}{b_{i}^{v}}}} & (1)\end{matrix}$

and are associated with each metabolite in the network. The shadowprices defined the sensitivity of the objective function (Z) to changesin the availability of each metabolite (b^(v) _(i) defines the violationof a mass balance constraint and is equivalent to an uptake reaction).The shadow prices were either negative, zero, or positive, depending onthe value of the metabolite. The direction and magnitude of the shadowprice vector in each region of the phase plane was different (bydefinition of the phase plane); thus, the state of the metabolic systemwas different in each region.

Isoclines: Isoclines were also defined to interpret the metabolicphenotype. Isoclines were defined to represent the locus of pointswithin the two-dimensional space that provide for the same value of theobjective function. The slope of the isoclines within each region wascalculated from the shadow prices; thus, by definition the slope of theisoclines was different in each region of the PhPP. A ratio of shadowprices was used to define the slope of the isoclines (ρ):

$\begin{matrix}{{{{{{\rho = {- \frac{\gamma_{x}}{\gamma_{y}}}}}_{Z} = {- \left( \frac{{- {Z}}/{b_{x}^{v}}}{{- {Z}}/{b_{y}^{v}}} \right)}}}_{Z} = {- \frac{b_{y}^{v}}{b_{x}^{v}}}}}_{Z} & (2)\end{matrix}$

The negative sign in Eqn. 2 was introduced in anticipation of itsinterpretation. The condition dependent relative value of thesubstrates, defined as ρ, was used to interpret the constraining factorson the metabolic network. In regions where ρ was negative, there wasdual limitation of the substrates. Under different condition, theisoclines were also either horizontal or vertical in certain phase planeregions, representing regions of single substrate limitation, the ρvalue in these regions was zero or infinite, respectively. Certainregions in the PhPP also had a positive ρ; these regions were termed“futile” regions, and increased uptake of one of the substrates had anegative effect on the objective function in these regions. Finally, dueto stoichiometric limitations, there were infeasible steady stateregions in the PhPP.

Line of Optimality: The line of optimality (LO) was defined as the linerepresenting the optimal relation between the two metabolic fluxescorresponding to the axis of the PhPP. For results presented herein,this line can be interpreted as the optimal oxygen uptake for thecomplete oxidation of the substrate in order to support the maximalbiomass yield.

These procedures were applied to the reaction list for E. coli K-12defined by (Edwards and Palsson, The Escherichia coli MG1655 in silicometabolic genotype: its definition, characteristics, and capabilities,Proc. Natl. Acad. Sci. U.S.A., 97(10):5528-33 (2000b)). It generates thetwo and three dimensional phase planes that are used in the examplesbelow.

EXAMPLE 3 Optimal Behavior of E. COli Under Defined Conditions

This Example shows that the strain used exhibited optimal aerobic growthusing acetate and succinate as primary substrates without adaptiveevolution.

The list of metabolic reactions that take place in E. coli K-12 M1655has been assembled (Edwards and Palsson (2000b)). Based on this list astoichiometric matrix was formulated. Using maximal uptake rates foroxygen (on the y-axis) and a carbon substrate (on the x-axis) aphenotypic phase plane was calculated using the procedures describedabove. Specifically two carbon sources were used, acetate and succinate.Then the calculated phase planes were used to determine the optimalgrowth conditions and a series of growth experiments were performed. Thecomputational (i.e. in silico) and experimental results were thencompared.

Acetate. Optimal growth performance on acetate was investigated insilico, and the predictions generated were compared to experimentaldata. The in silico study started with a phenotype phase plane (PhPP)analysis with the acetate and oxygen uptake rates defined as the axes ofthe two-dimensional projection of the flux cone representing thecapabilities of E. coli metabolism (FIG. 1). The flux cone is the regionof all admissible steady state metabolic flux distributions (for acomplete description of the flux cone see ref (Schilling et al.,Metabolic pathway analysis: basic concepts and scientific applicationsin the post-genomic era, Biotechnol. Prog., 15(3):296 (1999)).Furthermore, a three-dimensional projection of the flux cone with thegrowth rate defined as the third dimension was utilized (FIG. 1). The insilico analysis of the acetate-oxygen PhPP has been described in Example2 above. The optimal (with respect to cellular growth) relation betweenthe acetate and oxygen uptake rate, and this line is referred to as theline of optimality (LO).

The PhPP was used to analyze and interpret the operation of themetabolic network. For example, under oxygen limitations thecharacteristics of the metabolic network may be defined by region 2 ofthe PhPP (FIG. 1&2), where the acetate uptake rate exceeds the optimalrelation to the oxygen uptake rate. From FIG. 1, it can be seen that ifthe metabolic network were operating within region 2, the optimalcapability to support growth would be increased by reducing the acetateuptake rate to a point on the LO. A similar interpretation can be madefor points within region 1, with oxygen and acetate switching roles.Hence, metabolic flux vectors defining a point in region 1 or region 2would indicate inefficient utilization of the available resources. Thus,the in silico PhPP analysis led to the conclusion that if the regulationof the E. coli metabolic network has evolved to operate optimally tosupport growth with acetate as the sole carbon source, the relationbetween the acetate and oxygen uptake rate and the growth rate should bedefined by the LO (FIG. 1&2).

The relation between the acetate and oxygen uptake rates and the growthrate was experimentally examined by cultivating E. coli MG1655 onacetate minimal medium. The acetate uptake rate was experimentallycontrolled by changing the acetate concentration in the minimal media.The uptake rates of acetate and oxygen and the growth rate were measuredand the experimental points were plotted on the PhPP (FIGS. 1 and 2).The calculated optimal relation between the acetate and oxygen uptakerate was then compared to the experimental data (FIG. 1). Comparison ofthe experimental data to the in silico predictions indicated a 14%difference between the slope (0.91) of the linear regression line forthe experimental data and the slope (1.04) of the in silico defined LOfor aerobic growth on acetate minimal media.

The measured and calculated growth rates were plotted as thethird-dimension above the acetate-oxygen PhPP (FIG. 2). The color-codedsurface represents the three-dimensional projection of the flux cone. Inother words, the color-coded surface defines the solution space, and allfeasible steady state metabolic flux distributions are confined withinthe surface. Yes we can. The LO on the two-dimensional phase plane(FIG. 1) is a projection of the edge on the three-dimensional surface onto the x,y-plane (acetate uptake rate, oxygen uptake rate). Theexperimental data were plotted in the three-dimensional space (FIG. 2).To quantitatively visualize the proximity of the data points to the LOin three-dimensions, the in silico predictions and the experimental datawere projected onto each plane formed by the basis vectors.

The projection of the three-dimensional LO and the experimental datapoints onto the (x,y), (x,z), and (y,z) (x-axis: acetate uptake rate;y-axis: oxygen uptake rate; z-axis: growth rate) planes is indicated inFIGS. 3&4 respectively, where the quality of the linear regression isindicated by the correlation coefficient, and the data are compared tothe in silico predictions. The predicted and the observed metabolicfluxes (substrate and oxygen uptake rates and growth rate) for eachpoint were directly compared and the in silico predictions and had anoverall average error of 5.8%. At this point, we should note that theinformation used to reconstruct the metabolic network was obtainedindependent from the present experiments (Edwards and Palsson, (2000b)).The calculated PhPP represents a priori interpretation and prediction ofthe data obtained in the present study.

Succinate. The succinate-oxygen PhPP (FIG. 5) was more complicated thanthe acetate-oxygen PhPP. The succinate-oxygen PhPP (FIG. 5) had 4distinct regions of qualitatively distinct optimal metabolic networkutilization. Regions 1 and 4 of the succinate-oxygen PhPP were analogousto regions 1 and 2 of the acetate-oxygen PhPP. For example, it can beseen from FIG. 5 that the maximal growth flux for a flux vector inregion 4 can be increased if the succinate uptake is reduced to a pointdefined by the region 3,4 demarcation. Furthermore, from the PhPPanalysis, region 3 is defined as a single substrate limited region. Thesingle substrate limited region indicates that the succinate uptake ratehas little effect on the maximal growth flux in region 3, whereas theoxygen uptake rate has a positive effect on the growth rate.

Region 2 is defined as a dual substrate limited region, since in region2 the metabolic network can support an increased growth rate if thesuccinate uptake rate is increased. The in silico analysis shows thatthe cellular growth rate can be increased by operating the metabolicnetwork off of the LO in region 2, by implementing a partially aerobicmetabolism and the secretion of a metabolic by-product. The optimalmetabolic by-product was calculated to be acetate. The production of areduced metabolic by-product in region 2 however reduces the overallbiomass yield. Therefore, based on the PhPP analysis, it may be surmisedthat, if the regulation of the metabolic network evolved toward optimalgrowth with succinate as the sole carbon source, the metabolic networkwill operate with a flux vector along the LO. However, the growth ratecan be increased by moving the flux vector into region 2, thus we expectthat the network should only operate in region 2 when oxygen is limitedand succinate is plentiful if the stated hypothesis is true.

E. coli growth experiments on succinate minimal M9 media were performedto critically test the hypothesis given the above in silico analysis.Multiple batch cultures were grown at various succinate concentrationsand temperatures to span a range of succinate uptake rates. The aerationand agitation were held constant to maintain a consistent maximal oxygendiffusion rate in all the cultures. The succinate and oxygen uptakerates and the growth rate were measured separately for each independentgrowth experiment. The experimental data were then directly compared tothe in silico predictions (FIG. 5).

The experimental data points were consistent with the stated hypothesis:the flux vector consistently identified points along the LO for oxygenuptake rates below a critical value (˜18.8 mmole.g-DW-1·hr−1).Furthermore, the cultures that identified points along the LO producedlittle or no acetate as a metabolic by-product (as predicted by the insilico analysis—see FIG. 5). As hypothesized, the experimental dataindicates horizontal movement of the flux vector within region 2 for theexperimental systems that are oxygen limited but have plentifulsuccinate. The break point in the experimental data was determined tocorrespond to a maximal oxygen uptake rate of 18.8±0.5 mmole*gDW−1*hr−1.Flux vectors within regions 3 or 4 were never observed. Acetateproduction was measured for the cultures identified in region 2, and theacetate production is quantitatively compared to the in silicopredictions in FIG. 6.

The optimal growth rate surface was constructed over thesuccinate-oxygen PhPP, and the measured flux vectors fell near the edgeof the polytope that corresponded to the LO (FIG. 7). The flux vectorsalso identified a locus of points on the phase surface in region 2 witha constant oxygen uptake rate equal to the maximal oxygen uptake limitof the system. To quantitatively test the predictive capability of thein silico analysis and the in silico derived hypothesis, we employed apiecewise linear model to describe our hypothesis and the experimentallyobserved flux vectors. The piecewise linear model is defined as follows:we identified the locus of points defined by the flux vector for a rangeof succinate uptake rates and an oxygen uptake limit. Below the oxygenuptake limit, the locus of points lies along the LO, and above theoxygen uptake limit the locus of points lies along the phase surfacewith a constant oxygen uptake rate (the oxygen uptake limit). Based onthe piecewise linear model, the succinate uptake rate was used topredict the oxygen uptake rate and the growth rate, and the other twopermutations were also considered. From this analysis an overall averageerror between the in silico predictions and the experimental data was10.7%.

This Example shows that the strain used exhibited optimal aerobic growthusing acetate and succinate as primary substrates. No adaptive evolutionwas necessary to achieve this optimal performance.

EXAMPLE 4 Evolution of a Sub-Optimal E. Coli Strain to Optimality

This Example demonstrates that E. coli can undergo some phenotypicadaptation from a sub-optimal growth state to an optimal statedetermined in silico.

Glucose: The glucose-oxygen PhPP contains six distinct regions (FIG. 8)Like the succinate-oxygen PhPP, region 1 represents futile cycles andsub-optimal growth performance, whereas region 2 is characterized byacetate overflow metabolism. The two are separated by the LO.

As before, the cellular growth rate, OUR, and glucose uptake rate (GUR)were experimentally determined over a range of glucose concentrationsand temperatures. Most experimentally determined values for the GUR,OUR, corresponded to points on the LO or slightly in region 2 of thePhPP (FIG. 8), where the predicted acetate secretion was experimentallyobserved.

In three dimensions, the measured growth rates lie on the surface of thesolution space near the edge corresponding to the LO, but are nottightly clustered there (FIG. 9). We therefore kept the strain insustained exponential growth (16) over a 40-day period (about 750generations) using serial transfer under constant growth conditions todetermine whether the metabolic phenotype would evolve (FIGS. 10 and11). Fitness indeed increased, as shown by movement of the experimentalpoints parallel to the LO, but there was no qualitative change in thephenotype.

EXAMPLE 5 Evolution of a Sub-Optimal E. Coli Strain to Optimality

This Example demonstrates that E. coli can undergo significantphenotypic adaptation from a sub-optimal growth state to an optimalstate determined in silico.

Glycerol: The glycerol-oxygen PhPP consists of 5 regions with featuresresembling those seen in the PhPPs for acetate, succinate and glucose.In particular, a region with futile cycles (phase 1) is separated froman acetate overflow region (phase 2) by the LO.

The growth performance over a range of glycerol concentrations wasexperimentally determined as before. In sharp contrast to growth onmalate or glucose, however, the experimental values for growth werescattered throughout phase 1, far from the LO (FIG. 12) and the surfaceof optimality (FIG. 13). Unlike the other substrates examined, glycerolthus supports only sub-optimal growth of E. coli K-12.

As before, we therefore performed a long-term adaptive growthexperiment, this time using glycerol as the sole carbon source. Theoriginal strain was again kept in prolonged exponential growth for 40days by serial transfer (17), maintaining a temperature of 30° C., aglycerol concentration of 2 g/L, and sufficient oxygenation. Growthrate, glycerol uptake rate (GlUR) and OUR were determined every tendays.

A forty-day evolutionary path (E1) was traced in phase 1, eventuallyconverging on the LO (FIG. 14). During this period, the growth rate morethan doubled from 0.23 hr⁻¹ to 0.55 hr⁻¹ (FIG. 15). Further testing ofthe resulting evolved strain (which was frozen and stored) revealedhigher specific growth rates and biomass yields than the parentalstrain. All of the data obtained fell on or near the LO as it had on thefinal day of the long-term culture (FIGS. 16 and 17), indicating thatthe evolved strain had attained an optimal growth performance onglycerol consistent with predictions in silico. A second, independentadaptation experiment gave a similar but not identical evolutionarytrajectory (E2), converging near the same endpoint. E. coli cantherefore undergo significant phenotypic adaptation from a sub-optimalgrowth state to an optimal state determined in silico.

Optimal growth performance of bacteria thus appears to conform with thepredictions of in silico analysis. On some substrates, such as acetateand succinate, the cell may display optimal growth, whereas on otherssuch as glucose and glycerol it may not. In the latter case growthevolves towards a phase plane predicted optimal performance.

All of the references cited herein are incorporated by reference.Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1. A method for optimizing a function of a biochemical reaction networkin a cell comprising: (a) calculating optimal properties of abiochemical reaction network by applying a computational optimizationmethod to a list of reactions representing said biochemical reactionnetwork; (b) altering one or more reactions in said list of reactions inthe biochemical reaction network and re-computing the optimalproperties; (c) repeating (b) until an optimized function is reached;(d) constructing the genetic makeup of a cell to contain the biochemicalreactions which result from (c); (e) placing the cells constructed under(d) in culture under a specified environment, and (f) cultivating thecells as in step (e) for a sufficient period of time and underconditions to allow the cells to evolve to the optimized functiondetermined under (c), wherein the biochemical reaction network comprisesa comprehensive biochemical reaction network.
 2. The method of claim 1,wherein the biochemical network is a metabolic network.
 3. The method ofclaim 1, wherein the biochemical network is a regulatory network.
 4. Themethod of claim 2, wherein the cells are prokaryotic cells.
 5. Themethod of claim 2, wherein the cells are eukaryotic cells.
 6. The methodof claim 1, wherein the eukaryotic cells are yeast, fungal cells, animalcells or cell lines.
 7. The method of claim 1, wherein step (d)comprises altering one or more genes in the eukaryotic cell.
 8. Themethod of claim 7, wherein altering comprises introduction of a gene orgenes into the eukaryotic cell.
 9. The method of claim 7, whereinaltering comprises modification of an endogenous gene or genes in theeukaryotic cell.
 10. The method of claim 1, wherein the biochemicalreaction network comprises a substantially whole biochemical reactionnetwork.
 11. A method for optimizing production of a biochemical,comprising: (a) calculating optimal properties of a biochemical reactionnetwork for production of a biochemical by applying a computationaloptimization method to a list of reactions representing said biochemicalreaction network; (b) altering one or more reactions in said list ofreactions in the biochemical reaction network and re-computing theoptimal properties; (c) repeating (b) until an optimized function forproduction of said biochemical is reached; (d) constructing the geneticmakeup of a eukaryotic cell to contain the biochemical reactions whichresult from (c); (e) placing the cells constructed under (d) in cultureunder a specified environment, and (f) cultivating the cells as in step(e) for a sufficient period of time and under conditions to allow thecells to evolve to the optimized function determined under (c), whereinsaid cells produce said desired biochemical.
 12. The method of claim 11,wherein the biochemical network is a metabolic network.
 13. The methodof claim 11, wherein the biochemical network is a regulatory network.14. The method of claim 12, wherein the cells are prokaryotic cells. 15.The method of claim 12, wherein the cells are eukaryotic cells.
 16. Themethod of claim 11, wherein the eukaryotic cells are yeast, fungalcells, animal cells or cell lines.
 17. The method of claim 11, whereinstep (d) comprises altering one or more genes in the eukaryotic cell.18. The method of claim 17, wherein altering comprises introduction of agene or genes into the eukaryotic cell.
 19. The method of claim 17,wherein altering comprises modification of an endogenous gene or genesin the eukaryotic cell.
 20. The method of claim 11, wherein thebiochemical reaction network comprises a substantially whole biochemicalreaction network.